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Proteasomal Regulation of the Hypoxic Response Modulates Aging in C. elegans

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Science  29 May 2009:
Vol. 324, Issue 5931, pp. 1196-1198
DOI: 10.1126/science.1173507

Abstract

The Caenorhabditis elegans von Hippel–Lindau tumor suppressor homolog VHL-1 is a cullin E3 ubiquitin ligase that negatively regulates the hypoxic response by promoting ubiquitination and degradation of the hypoxic response transcription factor HIF-1. Here, we report that loss of VHL-1 significantly increased life span and enhanced resistance to polyglutamine and β-amyloid toxicity. Deletion of HIF-1 was epistatic to VHL-1, indicating that HIF-1 acts downstream of VHL-1 to modulate aging and proteotoxicity. VHL-1 and HIF-1 control longevity by a mechanism distinct from both dietary restriction and insulin-like signaling. These findings define VHL-1 and the hypoxic response as an alternative longevity and protein homeostasis pathway.

Loss of protein homeostasis is increasingly becoming recognized as an important contributor to several age-associated diseases and may play a causal role in aging (1, 2). A link between aging and protein homeostasis in the nematode Caenorhabditis elegans is supported by observations that increasing life span by reducing insulin and insulin-like signaling (ILS) or by dietary restriction (DR) also improves function in transgenic models of proteotoxic disease associated with aberrant protein aggregation (3, 4).

A primary cellular mechanism for degrading damaged proteins is the ubiquitin-proteasomal system, which involves covalent attachment of ubiquitin to target proteins before degradation. RNA interference (RNAi) knockdown of proteasome components reduces resistance to polyglutamine toxicity and shortens life span in C. elegans (5, 6), and we noted that proteasome inhibition led to accelerated paralysis in animals expressing a 35-residue polyglutamine repeat fused to yellow fluorescent protein (YFP) in body wall muscle cells (Q35YFP) (fig. S2). To further explore the relations between proteasomal function and protein homeostasis, we initiated an RNAi screen of known or predicted E3 ubiquitin ligases for altered resistance to polyglutamine toxicity (7) (table S1). Cullin-RING ubiquitin ligases (CULs) consist of multiple protein subunits that include a cullin protein, a RING finger protein, an adaptor protein, and a substrate recognition subunit (fig. S3) (8). Similar to proteasome inhibition, RNAi knockdown of genes encoding CUL1 or CUL2 core components accelerated paralysis in Q35YFP animals (fig. S3).

In contrast to knockdown of CUL core components, we identified an RNAi clone corresponding to a CUL2 substrate recognition subunit, VHL-1, that significantly delayed paralysis in Q35YFP animals (Fig. 1A). A similar increase in resistance to β-amyloid toxicity was also observed in response to vhl-1(RNAi) (Fig. 1B). VHL-1 is homologous to the mammalian von Hippel–Lindau tumor suppressor protein, which ubiquitinates the α subunit of the hypoxic response transcription factor, HIF-1 (9). Under normoxic conditions, ubiquitination of HIF-1 by VHL-1 represses the hypoxic response by targeting HIF-1 for proteasomal degradation (fig. S4). In order for VHL-1 to ubiquitinate HIF-1, HIF-1 must be hydroxylated by the EGL-9 prolyl hydroxylase (10). Similar to vhl-1(RNAi), egl-9(RNAi) also enhanced resistance to both polyglutamine (Fig. 1C) and β-amyloid toxicity (Fig. 1D). Noting prior correlation between resistance to proteotoxicity and increased life span, we next determined whether vhl-1 and egl-9 also modulate aging by measuring the effect of RNAi knockdown of vhl-1 or egl-9 on life span in the RNAi-sensitive rrf-3(pk1426) background. Animals maintained on either vhl-1(RNAi) or egl-9(RNAi) lived significantly longer than animals maintained on empty vector (EV) bacteria (Fig. 1, E and F).

Fig. 1

VHL-1 and EGL-9 modulate proteotoxic stress and life span. RNAi knockdown of vhl-1 significantly enhances resistance to (A) polyglutamine (polyQ) toxicity (P < 1 × 10–5) and (B) β-amyloid (Aβ) toxicity (P < 1 × 10–5) relative to animals fed EV bacteria. RNAi knockdown of egl-9 significantly enhances resistance to (C) polyglutamine toxicity (P < 1 × 10–5) and (D) β-amyloid toxicity (P < 1 × 10–5) relative to animals fed EV bacteria. RNAi knockdown of (E) vhl-1 (P < 1 × 10–5) or (F) egl-9 (P < 1 × 10–5) significantly increased adult life span relative to the EV-fed control. Paralysis and life-span statistics are in tables S1 and S6.

To determine whether increased stability of HIF-1 could account for the enhanced longevity associated with vhl-1 knockdown, we examined the life spans of animals deleted for vhl-1, hif-1, or both vhl-1 and hif-1 (10). The hif-1(ia4) allele removes exons 2, 3, and 4 of hif-1, including the DNA binding domain, and is believed to be a null allele (11) (Fig. 2A). The vhl-1(ok161) allele removes exons 1 and 2 of vhl-1 and is also a putative null allele (Fig. 2B). As observed for vhl-1(RNAi) animals, deletion of vhl-1 significantly increased life span (Fig. 2C). Deletion of hif-1 alone did not substantially influence life span but completely suppressed the life-span extension imparted by deletion of vhl-1 (Fig. 2C). Consistent with the observed longevity effects, the accumulation of autofluorescent age pigments, which has been proposed as a biomarker of aging and health span in C. elegans (12), was reduced in vhl-1(ok161) animals (Fig. 2D and fig. S5). This reduction was also fully suppressed by deletion of hif-1.

Fig. 2

VHL-1 modulates longevity, age-pigment accumulation, and reproduction in a HIF-1–dependent manner. Gene structures of (A) known hif-1 splice variants, hif-1(ia4) deletion, and hif-1(RNAi) and (B) known vhl-1 splice variants, vhl-1(ok161) deletion, and vhl-1(RNAi). Black boxes represent exons; yellow asterisk indicates a stop codon. Blue boxes indicate RNAi target sequences from Ahringer (Ah) or Vidal (Vi) library clones. The RNAi clones used to knock down hif-1 and vhl-1 target all known splice variants. (C) Vhl-1(ok161) animals are significantly longer-lived than wild-type (N2) animals (P < 1 × 10–5); vhl-1(ok161); hif-1(ia4) double-mutant animals are not longer-lived than N2 (P = 0.66). (D) Accumulation of autofluorescent age pigment is significantly reduced by deletion of vhl-1 (P < 1 × 10–5). Autofluorescence is not significantly different in N2 versus vhl-1(ok161); hif-1(ia4) double-mutant animals (P = 0.17). (E) Vhl-1(ok161) animals produce significantly fewer progeny than N2 animals do (P = 3.6 × 10–3). No significant difference in brood size was observed for vhl-1(ok161); hif-1(ia4) double-mutant animals (P = 0.69) or hif-1(ia4) animals (P = 0.43), relative to N2 animals. Data in (D) and (E) are mean ± SD of at least nine animals per condition. Life-span statistics provided in table S6.

Given that deletion of vhl-1 increased life span and resistance to proteotoxic stress, we speculated that there may be a fitness cost associated with constitutive expression of HIF-1 under normoxic conditions. One cost associated with many long-lived mutants is a decrease in fecundity. We quantified the number of eggs laid during adulthood (brood size) for N2, vhl-1(ok161), hif-1(ia4), and vhl-1(ok161); hif-1(ia4) animals. A significant decrease in brood size was observed for vhl-1(ok161) animals but not for hif-1(ia4) animals (Fig. 2E). As observed for life span and age-pigment accumulation, deletion of hif-1 suppressed the brood size defect of vhl-1(ok161) animals. Induction of HIF-1 by growth under hypoxic conditions also resulted in a significant decrease in brood size (figs. S6 and S7) and a corresponding increase in life span (fig. S8). These observations support the idea that repression of HIF-1 under normoxic conditions confers a fitness benefit in the form of enhanced fecundity.

We next examined the relations between DR and the hypoxic response. DR can be accomplished in C. elegans by reducing the availability of the bacterial food source, with complete removal of bacterial food during adulthood (bacterial deprivation) providing maximal life-span extension (13, 14). If vhl-1 and DR act in the same pathway to modulate longevity, then life-span extension from bacterial deprivation should require hif-1 and not further extend the life span of vhl-1 mutants. In contrast, bacterial deprivation extended the life span of hif-1(ia4) animals to an extent similar to that of controls (Fig. 3A) and further extended the long life span of vhl-1(ok161) animals (Fig. 3B). Bacterial deprivation also increased the life span of hif-1(ia4); vhl-1(ok161) double mutants (Fig. 3C).

Fig. 3

VHL-1 and HIF-1 modulate longevity by a mechanism distinct from dietary restriction. (A) Life-span extension from bacterial deprivation (BD) is not significantly different in N2 and hif-1(ia4) animals (P = 0.97). (B) BD significantly increases the life span of vhl-1(ok161) animals (P < 1 × 10–5) and (C) vhl-1(ok161); hif-1(ia4) double-mutant animals (P < 1 × 10–5). (D) Hif-1(RNAi) does not significantly alter the life-span extension of eat-2(ad465) animals (P = 0.6). (E) The eat-2(ad465) mutation significantly reduces pharyngeal pumping rate relative to rates in N2 (P < 1 × 10–5) or vhl-1(ok161) (P < 1 × 10–5) animals. Pharyngeal pumping rate is not significantly different in N2 and vhl-1(ok161) animals (P = 0.06). Data are mean ± SD. (F) Relative to animals fed EV bacteria under normoxic conditions, vhl-1(RNAi) under normoxia or growth on EV bacteria under hypoxia (hyp, 0.5% oxygen) failed to significantly increase autophagy, as indicated by the presence of LGG-1::GFP puncta (P = 0.6 and 0.5, respectively). Thirty-five animals per condition were imaged for EV and vhl-1(RNAi). Seven animals were imaged for hypoxia. Data are mean ± SEM. Life-span statistics provided in table S6.

A common genetic model of DR in C. elegans is mutation of eat-2, which results in decreased food consumption because of a defect in pharyngeal pumping (15). Unlike eat-2(ad465) mutants, vhl-1(ok161) animals did not display a significant reduction in pumping rate (Fig. 3E), and, similar to the case for bacterial deprivation, knockdown of hif-1 had no detectable effect on life-span extension from mutation of eat-2 (Fig. 3D). Knockdown of vhl-1 or growth under hypoxic conditions also failed to cause a significant increase in the abundance of autophagic vesicles (Fig. 3F and fig. S9), a phenotype reported to be required for life-span extension associated with DR (16, 17). Thus, DR and the hypoxic response are likely to modulate longevity via distinct genetic pathways.

Decreased activity of the insulin-like receptor DAF-2 has been shown to increase life span (18, 19) and promote resistance to hypoxia (20), leading us to consider whether vhl-1 and daf-2 act in the same genetic pathway to limit longevity. Like DR, however, daf-2(RNAi) further extended the already long life span of vhl-1(ok161) animals (Fig. 4A), and deletion of hif-1 (Fig. 4B) or both hif-1 and vhl-1 (Fig. 4C) did not prevent life-span extension from daf-2(RNAi). Life-span extension of animals with reduced ILS activity, including daf-2 mutants, is dependent on the FOXO family transcription factor DAF-16, which acts downstream of DAF-2 to regulate gene expression (21, 22). In order for DAF-16 to regulate target genes, it must be localized to the nucleus, a process that can be monitored by visualization of a DAF-16::GFP (green fluorescent protein) reporter (23). Transient heat shock or daf-2(RNAi) increased nuclear localization of DAF-16, whereas vhl-1(RNAi) had no detectable effect (Fig. 4D and fig. S10), suggesting that DAF-16 is not activated by loss of vhl-1. Consistent with this, daf-16(RNAi) did not fully suppress the increase in life span (Fig. 4E) or the reduced abundance of age pigment (Fig. 4F and fig. S11) associated with deletion of vhl-1, and vhl-1(RNAi) increased the life span of daf-16 null animals (fig. S12). In contrast, daf-16(RNAi) fully suppressed the enhanced longevity of daf-2(e1370) animals (fig. S12), further phenotypically differentiating deletion of vhl-1 from mutation of daf-2.

Fig. 4

ILS and VHL-1 modulate longevity by distinct mechanisms. Daf-2(RNAi) significantly increases the life span of (A) vhl-1(ok161) (P < 1 × 10–5), (B) hif-1(ia4) (P < 1 × 10–5), and (C) hif-1(ia4); vhl-1(ok161) animals (P < 1 × 10–5). (D) Vhl-1(RNAi) does not induce nuclear localization of DAF-16. Daf-2(RNAi) or heat shock significantly increases DAF-16 nuclear foci (P < 1 × 10–5 in each case). DAF-16 nuclear foci per animal was quantified for 10 animals per group. (E) Daf-16(RNAi) does not prevent life-span extension from deletion of vhl-1 (P < 1 × 10–5). (F) Deletion of vhl-1 significantly reduces autofluorescence in animals fed EV bacteria (P < 1 × 10–5) or daf-16(RNAi) (P = 0.004) but does not reduce autofluorescence in animals fed hif-1(RNAi) (P = 0.9). Median integrated pixel density shown for at least 10 randomly chosen animals per condition. Life-span statistics provided in table S6. Data in (D) and (F) are mean ± SEM.

Our data support a model in which vhl-1 and daf-2 modulate longevity by different mechanisms, but it remains possible that ILS and the hypoxic response act through an overlapping set of target genes (fig. S1). Multiple DAF-16 target genes appear to be important for life-span extension in response to reduced ILS (24), and we speculate that multiple HIF-1 target genes may contribute to life-span extension in vhl-1(ok161) animals, some of which may be shared with DAF-16. Microarray studies have indicated that HIF-1 and DAF-16 have shared target genes (25, 26), and mutation of daf-2 can lead to increased resistance to hypoxic stress (20). In addition, reduced ILS and hypoxic response both induce resistance to heat stress (27), a phenotype often correlated with longevity. Like DAF-2, VHL-1 acts postdevelopmentally to modulate life span by a mechanism distinct from DR; however, unlike the case for daf-2(e1370) animals, vhl-1(ok161) animals did not show an enhanced frequency of dauer formation (table S2), suggesting that, if shared downstream effectors modulate aging and protein homeostasis, they are separable from the DAF-16 target genes involved in dauer formation.

Several features of the hypoxic response are highly conserved from nematodes to mammals, including regulation of mammalian HIF-1 by VHL-1 and the identity of many HIF-1 target genes. This high level of conservation suggests that induction of the hypoxic response is likely to have many similar physiological effects in nematodes and humans. Although inappropriate activation of the hypoxic response can promote tumorigenesis, therapeutically targeting specific components of this pathway may prove useful for treating age-associated diseases in people, particularly disorders associated with proteotoxicity in postmitotic cells, such as Huntington’s disease, Alzheimer’s disease, and other neurological disorders.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1173507/DC1

Materials and Methods

Figs. S1 to S12

Tables S1 to S6

References

  • * These authors contributed equally to this work.

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. We would like to thank B. Kennedy and P. Kapahi for helpful discussion. Strains were provided by the Caenorhabditis Genetics Center. This work was supported by Alzheimer’s Association grant IIRG-07-60158, a Glenn/AFAR (American Federation for Aging Research) Breakthroughs in Gerontology award, and NIH grant 1R01AG031108-01 to M.K. R.M. and K.A.S. were supported by postdoctoral fellowships from the Hereditary Disease Foundation. G.L.S., L.S.S., and F.J.R. were supported by NIH grant P30AG013280. M.K. is an Ellison Medical Foundation New Scholar in Aging.
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